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Jim Wilson has been one of gene therapy's pioneers. Now he's raising concerns about the safety of a technology he helped develop – at least when it is pushed to its limits. In two new studies his team conducted in monkeys, high doses of a certain type of gene were toxic.

"I recognize the fact that these are limited numbers [of animals], and in fact that studies were not designed to evaluate safety," Wilson says in an interview with Forbes, reproduced below. "But the fact is, we observed the toxicity with that limited number, which is, from my point of view, even more worrisome."

For decades, gene therapy – treating or curing sick people by replacing the worst parts of their DNA – has been a pipe dream. Now, it's a reality. Last year, the Food and Drug Administration approved gene therapies for forms of rare blood cancer, and a gene therapy to treat blindness.

All gene therapies rely on what scientists call "vectors" – which just means a way of getting DNA into a patient's cells. The most common vectors? Viruses. Hijacking cells to produce copies of themselves, after all, is what viruses do. And Wilson, the Rose H. Weiss Orphan Disease Center Director's Professor at the University of Pennsylvania, is the authority on vectors.

It's a hard-won expertise. In 1999, when it seemed that gene therapy was on the cusp of becoming a reality, a patient in a study Wilson was running died. The memory of that young man, 18-year-old Jesse Gelsinger, has motivated him to find better vectors. Gone is the widespread use of adenovirus, the vector used in that study. Wilson has become an advocate for another virus, adeno-associated virus, or AAV, which is used in many gene therapies including the blindness treatment Luxturna, approved by Spark Therapeutics.

That's why it shocked the gene therapy field when Wilson resigned as a scientific advisor to one AAV-focused firm, Solid Biosciences, just before the company went public, noting concerns about the safety of high-dose AAV. He says he can't talk about his relationship with Solid, but has published twodifferent papers using two different versions of the AAV virus showing that it can have potentially lethal side effects used high doses in monkeys. For most uses (like blindness) these high doses aren't necessary. But for some conditions, like the muscle disorders Solid is targeting, they are.

Wilson spoke with Forbes at length about the results, and I'm publishing the bulk of the interview – edited slightly for length and clarity. "At the end of the day, the best we can do right now is to be as diligent as we can in non-clinical models to determine at what dose we would see benefit, and, similarly, at what dose we would see dose-limiting toxicity, and to select diseases in which the unmet need is so significant that it would justify the risks that, despite animal models, are largely unknown," he says. Read the interview below.

Matthew Herper: Before we get into the topic of this paper, let's talk a little bit about you. You've been a major player in this space. You've spoken very eloquently about the experience of treating Jesse Gelsinger, who died while being treated with gene therapy in 1999. How is that coloring the way you're thinking about the field now?

James Wilson: I've been focused on gene therapy for probably over 30 years. During that time, have assessed different vector technology platforms from those based on viruses and those based on non-viruses. What I did realize at the turn of the century, after the [ornithine transcarbamylase deficiency] trial and Jesse Gelsinger's death, is that we really needed better delivery vehicles, and that's what I focused my career on since. I believe that the technology of AAV that we now have is by far the best-in-class, and in the right circumstances, will be successful in treating patients.

MH: Let's get right to what you found, then, because you found what could potentially be a speed bump here in using that technology at least when you're using high doses of AAV.

JW: Most of the work that is underway in this field and almost all the work that we're conducting uses AAV vectors that are injected directly in the tissues or into certain compartments such as the retina or the central nervous system. There are a set of studies in the clinic that are based on injecting the vector into the blood after which it distributes broadly through many organs, but in particular, the liver. Those studies have formed the basis of some really exciting work in treating liver diseases such as hemophilia.

What we're talking about here, though, is an extreme example of the use of vectors to inject directly into the blood at very high doses in order to drive the vector into tissues that they otherwise do not penetrate, such as the skeletal muscle to treat inherited muscle diseases such as Duchenne muscular dystrophy, or into the brain to treat diseases such as spinal muscular atrophy.

We conducted two different animal studies in non-human primates and in pigs to study the efficacy of high-dose intravenous AAV to target the central nervous system. We did not expect to see any safety concerns because of the incredible safety track record that AAV has had. But what we indeed saw in both studies was that, at very high doses, the animals developed within the first few days after the vector was injected evidence for some liver damage and also activation systemically of inflammation, the consequences of which, was the development of a bleeding disorder or coagulopathy. What was surprising is this happened really in the first few days after the vector was administered. Now, in both studies, this resolved in some animals and they were fine. But in each case, one of the animals progressed to very significant disease manifested by shock, in one case, and severe hemorrhage or bleeding in the other that required euthanasia.

So this suggested that, selected at high enough doses injected in directly into the blood, that there is the potential for what we call dose-limiting toxicity. While we were surprised to see this because it hadn't been described, in retrospect it shouldn't be surprising because any biologic, if delivered at high-enough dose, will be associated with toxicity. I think what we've observed here is if that does occur, when injected at high dose, intravenously, these are the kinds of things that we would see. Therefore, these are the kinds of things that we should monitor for in the clinic.

MH: These are small numbers of animals. Couldn't this be the play of chance? Or could it be the animals that had the problems that had the coagulopathy or the shock were outliers in some way?

JW: I recognize the fact that these are limited numbers, and in fact that studies were not designed to evaluate safety. But the fact is, we observed the toxicity with that limited number, which is, from my point of view, even more worrisome.

In drug development, unfortunately, what often shuts down programs are the outliers. In this situation, really one out of three in the first study of the non-human primates, and three out of three of the piglets had the toxicity.

We've learned what the dose-limiting toxicities could be at high enough dose when injected intravenously. But what we have not learned is at what dose would this occur across multiple programs. I would caution anyone to directly extrapolate the dose at which we observe the toxicity in our study to that of another program.

Every lab measures the quantity of vectors used to dose differently. Those numbers can vary well over 10 fold. In other words, the monkey studies that were conducted at Audentes [Ed.: another biotech company] that support their gene therapy program in myotubular myopathy dosed the monkeys at eight times ten to the fourteenth copies per kilogram, which is a very high dose. The animals did fine and the company has proceeded with what appears to be the beginning of a successful clinical trial.

That does not mean that other vectors administered at that dose will be safe. On the flip side, if we saw toxicity at a dose of four times ten to the fourteenth copies per kilogram. That doesn't mean that you would expect to see toxicities at that dose in other programs as well.

MH: Why do you think these side effects occurring? You said you were surprised. Why are we getting these dose limiting toxicities? Do we have any idea of biologically what's happening?

JW: There are some data that the vector capsid, the shell of the viral genome itself, may be capable of activating what is called innate immunity. What we learned from the adenovirus trials was that it was the capsid of the shell that in some patients, under some circumstances, when injected systemically, would bind to immune-activating cells and immediately stimulate them to secrete inflammatory cytokines. In fact, our innate immune system has been designed to detect invaders and viruses, and the first response is the innate immune response. That's what I think we saw here.

MH: What was the specific goal of the study as planned? What were you testing?

JW: The first study in Human Gene Therapy was designed to evaluate efficiency of gene transfer in cells of the spinal cord and the brain using different routes of administration. There were a number of arms to the study that included directly injecting the vector into the cerebral spinal fluid, so-called intrathecal delivery using a variety of different approaches.

They were then compared to this arm of the study, which was to evaluate high dose systemic vector, which has been proposed by several groups as a way to get vectors delivered to the central nervous system. This was almost the control arm for the purpose of the study, which was to evaluate directly administering the vector into the brain.

The second study, in Molecular Therapy, was designed to evaluate the performance of a new vector that was published out of a lab in Cal Tech called PHPB, versus AAV9 for delivering genes to the central nervous system. In that paper, they described remarkable targeting to the brain with very low quantities of vector when administered intravenously in mice.

The goal of this study was to compare gene transfer to the brain based on an AV vector versus a PHP.B vector to determine whether the mouse data translated to monkeys. It didn't.

MH: But it sounds like you found the same dose limiting toxicity? What happened to those animals?

JW: Yes. In that study, we dosed animals at a modest dose, because they're monkeys, of AAV9 and PHP.B at doses that would be required to target the liver. But we did not see any enhanced gene transfer into the brain. Then we went to a higher dose close to that, but not quite, used in the first study. In there, the AAV9 animal had some lab abnormality but did okay.

But the PHP.B vector-treated animal developed within a few days the elevation in the liver enzyme and then the bleeding disorder or coagulopathy just like we had seen in the other animals. That animal had to be euthanized within the first five days of receiving vector.

MH: What would you advise companies -- or any researcher – to do differently now?

JW: Let me recognize that decisions to proceed into human studies are complicated. They're difficult decisions that need to be informed by the potential for risks that we have to concede are largely unknown, reconciled against the unmet need of the patients and the likelihood of benefit.

At the end of the day, the best we can do right now is to be as diligent as we can in non clinical models to determine at what dose we would see benefit, and, similarly, at what dose we would see dose-limiting toxicity, and to select diseases in which the unmet need is so significant that it would justify the risks that, despite animal models, are largely unknown.

MH: Is it possible that we could prevent these toxicities in patients, while still allowing them to try these experimental therapies?

JW: That's a really good question because I think it's an area that would definitely deserve some investigation. If our hypothesis is right, what we're seeing is the immediate activation of innate immunity. It reminds me of what happened with the CAR-T study recently where there was this sort of mass influx of T-cells, and activation of immunity, a systemic inflammatory response syndrome that had the potential to be lethal. That could have shut the program down, which would have been very unfortunate.

A group of investigators and various different labs determined that the way to prevent this from happening is, at the time the cells are administered, the patients are treated with an antibody to try to suppress the consequences of the inflammatory response.

If these kinds of toxicities begin to surface in the clinic, it would warrant studies to try to mitigate them through the administration of drugs at the time the vector is delivered to try to dampen or to suppress the inflammatory response.

Currently in the field, many clinical trials are including a short-term dose of corticosteroids at the time of vector administration. My hope is that there may be a cocktail of immune modulating drugs that could be administered at the time the vector is delivered.

MH: Could the toxicities be due to impurities in the vector preparation or anything like that?

JW: You can never rule out a contaminant contributing to an outcome when the product is made by cells. But the reason that I think it's unlikely in our experiment is that we saw the same kind of toxicity at similar doses in two studies in which the method of vector production and purification were very different.

MH: Why was there a difference in the toxicity between species?

JW: Our experience with vector-host interaction over the years with viral and non-viral vectors shows dramatic differences in the innate immune response or inflammatory response between animals. It's very hard to see in rodents, more severe in cats and dogs, and always the most extreme in primates. We have very limited experience in pigs, so I really don't know enough as to why the systemic and liver toxicity was not present in pigs versus non-human primates.

MH: Coming at this from another direction: why weren't these studies of dose limiting toxicity in these vectors done in primates until now? Normally, one of the things that you worry about a lot before a phase one trial starts is exactly this. It's finding that dose limiting toxicity.

JW: We've conducted studies in thousands of non-human primates, probably more so than almost the rest of the field. But we've never pushed the dose to these limits, so we would not have seen them before, based on the kind of work that we do.

Only a few programs, very few, are progressing their programs along a path of high dose systemic AAV. I suspect that the amount of experience at these doses is really low. Let me tell you another practical issue. It takes a lot of resources to make that vector. They're not experiments that are easy to do from a logistical or financial standpoint.

MH: Right. And, as you said, this doesn't relate to the hemophilia trials. It doesn't relate to the eye trials.

JW: The fact is that not many sponsors have proceeded down this path of very high dose vectors to target muscle or the brain. And it's not something that one would do unless you needed to create to a potential therapeutic. There's a limited database.

My view, and this is an hypothesis, is that any AAV factor, if pushed at high enough dose, is going to start to surface dose-limiting toxicity as we talked about. At which dose and where that will occur will vary from product to product.

We're really early in this game. I suspect others are going to begin to study this as we will begin to try to parse out what the mechanisms are. Understanding mechanisms even at the extreme can help possibly mitigate other problems that may occur. Maybe not with such dire consequences, but having said that, to do those studies, you need a lot of vector and you need access to non-human primates.

MH: We have human data in some of these dose indications, right. The AveXis product has been tried in humans. So far, we haven't seen these toxicities. Does that mean anything to you?

JW: I think it's a really important context that in the AveXis trial that they progressed 12 subjects, maybe more now, at those doses. That's encouraging for those programs. I would agree.

MH: Is there anything else you'd add here?

JW: In my 30-plus years in this field, I studied every viral vector that has been proposed, and early in my career and more recently, non-viral vector.

The safety profile of AAV with respect to immune toxicity is remarkable and qualitatively better than the experiences that I've had with other delivery platforms. That has not changed in my mind at all.

Most of the applications that are being contemplated, I still expect there to be a pretty good safety profile. However, if the doses get too high, I believe that there is the potential for dose limiting toxicity. I don't think anyone should be surprised by that. But let's not impugn the platform for what it is.

I believe this is biology's century. I've covered science and medicine for Forbes from the Human Genome Project through Vioxx to the blossoming DNA technology changing

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I believe this is biology's century. I've covered science and medicine for Forbes from the Human Genome Project through Vioxx to the blossoming DNA technology changing the world today.
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